Mechanisms of Small Heat Shock Proteins

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Maria K. Janowska, Hannah E.R. Baughman, Christopher N. Woods, and Rachel E. Klevit

Department of Biochemistry, University of Washington, Seattle, Washington 98195 Correspondence: [email protected]

Small heat shock proteins (sHSPs) are ATP-independent chaperones that delay formation of harmful protein aggregates. sHSPs’ role in protein homeostasis has been appreciated for decades, but their mechanisms of action remain poorly understood. This gap in understanding is largely a consequence of sHSP properties that make them recalcitrant to detailed study. Multiple stress-associated conditions including pH acidosis, oxidation, and unusual availability of metal ions, as well as reversible stress-induced phosphorylation can modulate sHSP chaperone activity. Investigations of sHSPs reveal that sHSPs can engage in transient or long- lived interactions with client proteins depending on solution conditions and sHSP or client identity. Recent advances in the field highlight both the diversity of function within the sHSP family and the exquisite sensitivity of individual sHSPs to cellular and experimental conditions. Here, we will present and highlight current understanding, recent progress, and future challenges.

lthough small heat shock proteins (sHSPs) et al. 2012). sHSPs are implicated in muscle pro- Awere recognized as protein chaperones a tection, their expression is associated with poor quarter century ago (Horwitz 1992; Jakob et al. prognosis and treatment resistance in cancer, 1993), understanding how they work at a molec- and they play ameliorative roles in Parkinson’s ular level has been slow to emerge. sHSPs are and Alzheimer’s disease (Zoubeidi and Gleave deﬁned by their shared α-crystallin domain 2012; Dubińska-Magiera et al. 2014; Leak (ACD), named after the highly abundant sHSPs 2014). Transcription of some, but not all, sHSPs in the eye lens, αA-crystallin (referred to here is under the control of the heat shock fac- by its gene name, HSBP4) and αB-crystallin tor (HSF) transcription factors, which can up- (HSPB5) (Caspers et al. 1995). sHSPs are ATP- regulate the cellular concentrations of an sHSP independent chaperones that can delay the onset in response to stress (Table 1) (De Thonel et al. of irreversible protein aggregation in response to 2012; Zhong et al. 2016). In addition, sHSPs cellular stressors. Mutations in sHSPs are linked themselves are exquisitely sensitive to their con- to multiple diseases, including various neurop- ditions, and their activity, as well as their protein athies and early-onset cataract formation, im- levels, is activated by cellular conditions (Hasl- plying that sHSP dysfunction can have dire con- beck et al. 2005; Treweek et al. 2015). Genomes sequences (Litt et al. 1998; Vicart et al. 1998; across biology contain varying numbers of Irobi et al. 2004; Kijima et al. 2005; Hansen sHSPs: Escherichia coli and Saccharomyces cer- et al. 2007; Houlden et al. 2008; Datskevich evisiae each have two; Drosophila melanogaster

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M.K. Janowska et al.

Table 1. Basic information regarding human small heat shock proteins (sHSPs) Gene name Other names Tissue distribution Heat shock factor (HSF) inducible HSPB1 Hsp25, Hsp27, Hsp28 Ubiquitous HSF-1, HSF-2 HSPB2 MKBP Cardiac and skeletal muscle HSPB3 HSPL27 Cardiac and skeletal muscle HSPB4 αA-crystallin Eye lens HSPB5 αB-crystallin Ubiquitous HSF-1 HSPB6 Hsp20, p20 Ubiquitous HSPB7 cvHsp Cardiac and skeletal muscle HSPB8 Hsp22 Ubiquitous HSF-1 HSPB9 CT51 Testis HSPB10 ODF1 Testis

has 12; Caenorhabditis elegans has 16; and terminal region (NTR) and a flexible carboxy- Arabidopsis thaliana has 25 (Susek and Lind- terminal region (CTR) flank the structured ACD quist 1989; Laskowska et al. 1996; Wotton et al. (Fig. 1). The three domains show distinct be- 1996; Scharf et al. 2001; Candido 2002; Michaud haviors that arise, at least in part, from their et al. 2002). The ten human sHSPs differ in their distinct amino acid content. The ACD is the tissue distribution and response to specific only natively folded region, forming an IgG- stressors (see Table 1) (Kappé et al. 2003). like β-sandwich structure (Fig. 1B). ACDs of hu- Recent studies have begun to define how man sHSPs are enriched in histidines that may sHSPs become activated and how they recognize give rise to an ability to respond to changes in “clients” (the proteins on which they act) (Mainz pH and in metal ion availability to modulate et al. 2012; Peschek et al. 2013; Rajagopal et al. sHSP activity (Fig. 1A). The CTR is enriched 2015b). Such studies are proving to be highly in polar and charged residues, is highly disor- informative, although their interpretation in dered, and is thought to serve as a solubility tag terms of general models for sHSP activity has to enable the extremely high concentrations of proven challenging. As sHSPs act as early re- sHSP found in tissues such as eye lens (>150 mg/ sponders to help cells cope with proteins that mL) to remain soluble (Smulders et al. 1996; are destabilized because of a stress condition, Horwitz 2003). NTRs are enriched in hydropho- the “client-ome” could be vast, diverse, and dif- bic residues and are disordered (Bloemendal ferent depending on the state of the cell before 1977). Despite their simple architecture, sHSPs the stress. The sheer diversity of potential clients are structurally complicated. Human sHSPs ex- makes it unclear whether results obtained on ist in a range of oligomeric states. Some, includ- specific systems can be generalized to other ing HSPB1, HSPB4, and HSPB5, form polydis- sHSP systems and whether it is sensible to try perse ensembles of oligomers that range in size to do so. Here, we discuss emerging models and from dimers to ∼40-mers (Aquilina et al. 2003; outstanding questions regarding sHSP mecha- Horwitz 2003; Jovcevski et al. 2015). These olig- nisms and suggest strategies to leverage both omeric ensembles are highly dynamic with fre- technological and scientific developments to im- quent subunit exchange between oligomers (Pe- prove understanding of these enigmatic but crit- schek et al. 2013). Other sHSPs, such as HSPB8 ical proteins. and HSPB6 exist predominantly as small oligomers or dimers (Bukach et al. 2004). To date, there is no evidence of an sHSP that exists pre- sHSP STRUCTURE dominantly as a monomer, but such species may exist fleetingly as subunits exchange from one sHSPs Have Unusual Structural Properties oligomer to another (Bova et al. 1997). Like all sHSPs, the human sHSPs share a domain Although oligomeric mammalian sHSPs are architecture in which a highly variable amino- recalcitrant to conventional structural biology

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Mechanisms of Small Heat Shock Proteins

HSPB1 1 ----MTERRVP-FS-LLRGPSWDPFRDWYPHSRLFDQAFGLPRLPEEWSQWLG------GSSWPG------YVRPLPPA 61 HSPB2 1 ----MSGRSVP-HA----HPAT-AEYEFANPSRLGEQRFGEGLLPEEIL--TP------TLYHGY------YVRP---- 51 HSPB3 1 ----MAKIILR-HL------IEIPVRYQEEFEARGLEDCRLDHALY------ALPGPT------IVDL---- 45 HSPB4 1 -----MDVTIQ-HP-WFKRTLG-P---FY-PSRLFDQFFGEGLFEYDLLPFLS------STISPY------YRQ----- 50 HSPB5 1 -----MDIAIH-HP-WIRRPFF-P---FHSPSRLFDQFFGEHLLESDLFP-TS------TSLSPF------YLRP---- 51 HSPB6 1 -----MEIPVPVQPSWLRRASA-PLPGLSAPGRLFDQRFGEGLLEAELAALCP------TTLAPY------YLRA---- 57 HSPB7 1 -----MSHRTS-STFRAERSFH-SSSSSSSSSTSSSASRALPAQDPPMEKALSMF-----SDDFGS------FMRP---- 58 HSPB8 1 MADGQMPFSCH-YPSRLRRD---PFRDSPLSSRLLDDGFGMDPFPDDLTASWPDWALPRLSSAWPG------TLRS---- 66 HSPB9 1 ------MQRVG-NT------FSNESRVASRCPSVGLAERNRVATMP------VRLL--- 37 HSPB10 1 ----MAALSCL-LD-SVRRDIKKVDREL-RQLRCIDEFSTRCLCDLYMHPYCC------CDLHPYPYCLCYSKRSRSCGLCDLYPCC 74 Sip1 1 -----MSSLCP-YT------GRPTGLFRDF------EDMMPY------WAQR---- 28

HSPB1 62 AIESPAVAAPAYSRALSRQLSSGV-----SEIRHTADRWRVS------LDVNHFAPDELTVKTKDGVVEITGKHEERQDEHG 132 HSPB2 52 ------RAAPAGEG--SRAGA-----SELRLSEGKFQAF------LDVSHFTPDEVTVRTVDNLLEVSARHPQRLDRHG 111 HSPB3 46 ------RKTRAAQSPPVDSAAE----TPPREGKSHFQIL------LDVVQFLPEDIIIQTFEGWLLIKAQHGTRMDEHG 108 HSPB4 51 ------SLFRTV---LDSGI-----SEVRSDRDKFVIF------LDVKHFSPEDLTVKVQDDFVEIHGKHNERQDDHG 108 HSPB5 52 ------PSFLRAPSW-FDTGL-----SEMRLEKDRFSVN------LDVKHFSPEELKVKVLGDVIEVHGKHEERQDEHG 112 HSPB6 58 ------PSVALPV------AQVPTDPGHFSVL------LDVKHFSPEEIAVKVVGEHVEVHARHEERPDEHG 111 HSPB7 59 ------HSEPLAFPA-RPGGA-----GNIKTLGDAYEFA------VDVRDFSPEDIIVTTSNNHIEVRA---EKLAADG 116 HSPB8 67 ------GMVPRGPTATARFGVPAEGRTPPPFPGEPWKVC------VNVHSFKPEELMVKTKDGYVEVSGKHEEKQQEGG 134 HSPB9 38 ------RDSPAAQ------EDNDHARDGFQMK------LDAHGFAPEELVVQVDGQWLMVTGQQQLDVRDPE 91 HSPB10 75 LCDYKLYCLRPSLRSLERKAIRAIEDEKRELAKLRRTTNRILASSCCSSNILGSVNVCGFEPDQVKVRVKDGKVCVSAERENRYDCLG 162 Sip1 29 ------HSMLNNFNNIVPQQL-----NEVENTAQKFCVK------LDVAAFKPEELKVNLEGHVLTIEGHHEVK-TEHG 89

HSPB1 133 YISRCFTRKYT---LPPGVDPTQVSSSLSPEGTLTVEAPMPK--LATQSNEITIPVTFESRAQLGGPEAAKSDETAAK------205 HSPB2 112 FVSREFCRTYV---LPADVDPWRVRAALSHDGILNLEAPRGGRHLDTEVNEVYISLLPAPPD----PEEEEEAAIVEP------182 HSPB3 109 FISRSFTRQYK---LPDGVEIKDLSAVLCHDGILVVEVK------DPVGTK------150 HSPB4 109 YISREFHRRYR---LPSNVDQSALSCSLSADGMLTFCGPKIQTGLDATHAERAIPVSREEKP------TSAPSS------173 HSPB5 113 FISREFHRKYR---IPADVDPLTITSSLSSDGVLTVNGPRKQ----VSGPERTIPITREEKPAV------TAAPKK------175 HSPB6 112 FVAREFHRRYR---LPPGVDPAAVTSALSPEGVLSIQAA------PASAQAPP------PAAAK------160 HSPB7 117 TVMNTFAHKCQ---LPEDVDPTSVTSALREDGSLTIRARRHP------HTEHVQQTFRTEIKI------170 HSPB8 135 IVSKNFTKKIQ---LPAEVDPVTVFASLSPEGLLIIEAPQVP------PYSTFGESSFNNELPQDSQEVTCT------196 HSPB9 92 RVSYRMSQKVHRKMLPSNLSPTAMTCCLTPSGQLWVRGQCVA------LALPEAQTGPSPRLGSLGSKASNLTR------159 HSPB10 163 SKKYSYMNICKEFSLPPCVDEKDVTYSYGLGSCVKIESPCYPCTSPCSPCSPCSPCNPCSPCNPCSPYDPCNPCYPCGSRFSCRKMIL 250 Sip1 90 FSKRSFTRQFT---LPKDVDLAHIHTVINKEGQMTIDAPKTG----SNTTVRALPIHTSAGHAV--TQKPSSTTTTGK------158

B β C 4 β6+7 β8 β5 β3 β9

β3 β9

β8 β5 β4 β6+7

HSPB1: PDB 2N3J HSPB5: PDB 3J07 HSPB5: PDB 2YGD

Figure 1. General structural features of small heat shock proteins (sHSPs). (A) Sequence alignment of the 10 human sHSPs and Sip 1, a pH-sensitive sHSP from Caenorhabditis elegans generated with clustal omega and visualized with Jalview. The colored bar above the sequences identify the three regions of sHSPs: amino-terminal region (NTR) (blue), α-crystallin domain (ACD) (gray), and carboxy-terminal region (CTR) (red). Important sequence elements are highlighted in different colors:the conserved amino-terminal sequence (yellow), theβ4 and β8 strands that compose the groove (gray), the β6 + 7 strand that makes the dimer interface (green), and amino- and carboxy-terminal I/V-X-I/V motifs (blue). Histidine residues are highlighted in orange. The positions with sidechains pointing into the β4/β8 groove are indicated by purple dots, and the site of the “bump mutation” located in β8 is additionally labeled by a green bar crossing the purple dot. (B) ACD architecture. All sHSPs contain a core conserved ACD with an IgG-like β-sandwich fold. Human ACDs form an antiparallel dimer along the β6+7 strand. (C) Oligomeric organization of HSPB5. Two pseudo-atomic models of HSPB5 oligomers generated using a combination of solid-state nuclear magnetic resonanace (NMR), electron microscopy (EM), small-angle X-ray scattering, and structural modeling (left) (Jehle et al. 2011) and NMR, EM, and structural modeling (right) (Braun et al. 2011). Both depict 24-mers with tetrahedral geometry and extensive interactions among the three regions.

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M.K. Janowska et al.

approaches, truncated forms of sHSPs that con- sheet and a β8 strand from the “top.” Many tain an ACD are amenable to X-ray diffraction sHSPs contain a three-residue motif known as and nuclear magnetic resonanace (NMR). A “I/V-X-I/V” (i.e., isoleucine or valine, followed growing database of atomic-level structures of by any amino acid [usually a proline], followed ACDs reveal a common and highly similar sub- by isoleucine or valine), in their CTR, and bind- unit fold (Table 2; Fig. 1B). Small differences ing of this motif into the β4–β8 groove has been exist among available structures, but variation observed in crystal structures of HSPB1, HSPB4, in experimental conditions used makes it difﬁ- and HSPB5 (Table 2; Fig. 2A). In solution, the cult to draw functional insight from the differ- CTR of HSPB5 exists in equilibrium between ences. Nevertheless, clear common features are ACD-bound and unbound states (Jehle et al. revealed by these structures. Oligomerization of 2011; Baldwin et al. 2012; Delbecq et al. 2012). sHSPs is driven by a hierarchy of interactions. The CTR/ACD interaction plays an important Two subunits form a dimer through their ACD role in subunit recruitment both in the case of β-sandwich structures via antiparallel alignment HSPB5 homo-oligomers and HSPB5/HSPB6 of their long β6 + 7 strands (Fig. 1B). The “bot- hetero-oligomers (Delbecq et al. 2015) and likely tom slice” of the β-sandwich is composed of six in other sHSPs as well. More recently, it has be- β-strands, that is, β4, β5, and β6+7 fromeach come appreciated that I/V-X-I/V-like sequences subunit. Each ACD contains a hydrophobic appear in many NTRs (Fig. 1A). A crystal struc- groove at the opposite edge from the dimer in- ture of an HSPB2/HSPB3 heterotetramer reveals terface formed by a β4 strand from the “bottom” I/V-X-I/V motifs in the NTR of HSPB3 and the

Table 2. Mammalian small heat shock protein (sHSP) structures available in the Protein Data Bank (PDB) Ligand in β4/ PDB ID Protein Organism Method β8 groove? Mutations, notes 2N3J HSPB1 Human snNMR - 3Q9P HSPB1 Human X-ray - Atypical dimers; EE125/126AA 3Q9Q HSPB1 Human X-ray - Atypical dimers; EE125/126AA 4MJH HSPB1 Human X-ray Yes 6F2R HSPB2/HSPB3 Human X-ray Yes 3N3E HSPB4 Zebraﬁsh X-ray Yes 3L1F HSPB4 Bovine X-ray - 3L1E HSPB4 Bovine X-ray - Zn2+-bound 2KLR HSPB5 Human ssNMR - 2N0K HSPB5 Human snNMR - N146D 2WJ7 HSPB5 Human X-ray - 2Y1Y HSPB5 Human X-ray Yes L137MSE 2Y1Z HSPB5 Human X-ray Yes R120G, L137M 2Y22 HSPB5 Human X-ray - L137MSE 2YGD HSPB5 Human EM Yes S65T, Y66W 3J07 HSPB5 Human ssNMR; SAXS; EM Yes 3L1G HSPB5 Human X-ray - 4M5S HSPB5 Human X-ray Yes 4M5T HSPB5 Human X-ray Yes E117C 2WJ5 HSPB6 Rat X-ray - 4JUS HSPB6 Human X-ray Yes 4JUT HSPB6 Human X-ray Yes EE104/105AA 5LUM HSPB6 Human X-ray Yes 5LTW HSPB6 Human X-ray Yes Complex with 14-3-3/Ni2+ bound X-ray, X-ray diffraction; snNMR, solution-state nuclear magnetic resonance; ssNMR, solid-state nuclear magnetic resonance; EM, electron microscopy; SAXS, small-angle X-ray scattering.

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Mechanisms of Small Heat Shock Proteins

ABV183 I161 β β8 β 8 I181 I159 8 V7 V5

β4 β4 β4 HSPB1: PDB 4MJH HSPB5: PDB 4M5S HSPB6: PDB 5LTW

C β3 R27 β3 D

Q31

β6+7 β5

HSPB6: PDB 5LTW HSPB6: PDB 5LTW

Figure 2. Structural aspects of small heat shock protein (sHSP) assembly. Assembly of sHSP oligomers is driven by interactions that involve all three regions: the α-crystallin domain (ACD) (gray), the amino-terminal region (NTR) (blue), and the carboxy-terminal region (CTR) (red). PDB IDs are indicated below each panel. (A)ACD β4/β8 groove interactions with carboxy-terminal I/V-X-I/V motifs. The CTRs of many human sHSPs contain I/V-X-I/V motifs, which can dock into the β4/β8 groove on the outer edge of the ACD. Shown here, HSPB1 and HSPB5 ACDs were crystallized with peptides containing their carboxy-terminal I/V-X-I/V motifs (Hochberg et al. 2014). (B)ACDβ4/β8 groove interactions with an amino-terminal I/V-X-I/V motif. Several human sHSPs contain I/V-X-I/V motifs in their NTRs, which can also bind the β4/β8 groove. A crystal structure of full-length HSPB6 revealed its amino-terminal VPV motif bound in the groove (Sluchanko et al. 2017). (C) ACD dimer interface interactions with an NTR sequence. The HSPB6 structure revealed interactions between the conserved NTR sequence 27RLFDQ31 and a groove at the dimer interface of the ACD (Sluchanko et al. 2017). (D) Phos- phorylation-dependent NTR–client interactions. Phosphorylated Ser16 (teal) in the HSPB6 NTR facilitates interactions with the client protein 14-3-3 (orange). This may serve as a model for other phosphorylation- dependent client interactions and provides the only atomic-level information about NTR–client interactions available to date (Sluchanko et al. 2017).

CTR of HSPB2 bound in the β4–β8 grooves of in oligomerization to include the NTR and relax different HSPB2 subunits (Clark et al. 2018). An the deﬁnition of the I/V-X-I/V motif. I/V-X-I/V motif at the amino-terminal end of A structure of full-length HSPB6 revealed HSPB6 is inserted in the groove in a structure another NTR/ACD interaction in which the of full-length, phosphorylated HSPB6, an sHSP NTRsequence 27RLFDQRFG34 bindsinagroove that does not contain an I/V-X-I/V motif in its formed by the ACD dimer interface (Fig. 2C) CTR (Fig. 2B) (Sluchanko et al. 2017). A struc- (Sluchanko et al. 2017). This NTR sequence is ture of a truncated form of HSPB6 lacking the the sole conserved region among human NTRs NTR I/V-X-I/V motif revealed ﬁve ways in (Fig. 1A). In light of strong sequence conserva- which other hydrophobic residues such as leu- tion of the dimer interface, it is tempting to pre- cine and proline in the NTR can insert into the dict that other sHSPs may use an analogous β4–β8 groove (Weeks et al. 2014). Although NTR–ACD contact. Intriguingly, a recent struc- some of the observed interactions may be caused ture of an HSPB2/HSPB3 heterotetramer shows by crystal packing effects, as suggested by the a fragment docked in the ACD dimer interface investigators, the observation of knob-and-hole groove, but the data do not permit unambiguous interactions involving noncanonical I/V-X-I/V assignment of a protein segment to the electron motifs strongly suggests that we should expand density (Clark et al. 2018). Clarity on this point our view of the role of β4–β8 groove interactions must await further experimentation and, possi-

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bly, new structures. Models of HSPB5 oligomers state NMR, Mainz et al. (2015) showed that based on solid-state NMR and electron micros- HSPB5 uses different regions to bind different copy (EM) data depict extensive NTR–NTR in- clients. Lysozyme, which forms amorphous ag- teractions between adjacent protomers (Fig. 1C); gregates, is bound by the NTR of HSPB5, and but these interactions are not well-defined be- deletion of this domain abolishes HSPB5 chap- cause of the difficulty of characterizing disor- erone activity toward this client. The amyloid dered, heterogeneous protein regions (Braun et fibril-forming peptide Aβ1-40 binds in the hy- al. 2011; Jehle et al. 2011). drophobic β4–β8 groove of HSPB5 ACD, and Conservation of the interaction motifs de- chaperone activity is retained on deletion of scribed above implies that most sHSPs use these the NTR. An ACD dimer that lacks the NTR types of interactions to achieve their quaternary and CTR can inhibit aggregation of Aβ1-42, structures. However, the different oligomeric α-lactalbumin, and κ-casein, implying a direct propensities among human sHSPs suggest that role for this domain in HSPB5 chaperone activ- the relative contributions of each type of inter- ity for these clients (Hochberg et al. 2014). How- action differ and that structural variability is en- ever, although the chaperone activity of the ACD coded in each sequence. Furthermore, sHSP matches that of full length HSPB5 in the case of oligomeric structures are not static and are Aβ1-42 and α-lactalbumin in this study, it exquisitely responsive to environmental con- showed diminished chaperone activity against ditions, likely through modulation of the inter- κ-casein relative to full-length HSPB5, implying actions and their relative contributions (Jehle that additional regions are involved. Intriguing- et al. 2011; Peschek et al. 2013; Rajagopal et al. ly, HSPB5 ACD had enhanced chaperone activ- 2015b). ity relative to full-length HSPB5 toward the cli- Although information on sHSP structures ents α-synuclein and tau, leading to the proposal has increased in recent years, progress has been that the other domains may inhibit chaperone far slower than in most fields in which structural activity of the ACD (Liu et al. 2018). The HSPB1 biology has played a role (Table 2). The inherent NTR is vital for binding T4 lysozyme (Mc- properties of sHSPs pose substantial challenges Donald et al. 2012), and the isolated HSPB1 to the goal of defining their structures at atomic- ACD appears unable to bind fibrils composed level detail. Indeed, the dynamics and polydis- of the client protein α-synuclein (Cox et al. persity displayed by sHSPs beg the question of 2018). In the only structure of a full-length sHSP what it even means to “define” their structure. in complex with a binding partner currently We are optimistic that emerging approaches available, the NTR of phosphorylated HSPB6 including cryoelectron microscopy (cryo-EM), makes direct contact with the signaling protein solid-state NMR, and single-molecule tech- 14-3-3 (Sluchanko et al. 2017) via a phosphory- niques have the potential to greatly enhance lated serine in the HSPB6 NTR, which interacts our understanding of sHSP structures. directly with the client (Fig. 2D). In sum, available results implicate both the NTR and ACD in client recognition and chaperone activity, with Structural Features of sHSP/Client varying roles that depend on the client protein. Interactions To date, the CTR has not been shown to directly Despite the experimental challenges of defin- interact with any client protein, but its roles in ing sHSPs structurally, information regarding oligomerization and subunit exchange likely client-binding sites is beginning to emerge. A contribute to sHSP chaperone activity. combination of biochemical and structural ap- Natively folded proteins may be destabi- proaches has proven useful in elucidating the lized by changes in temperature, pH, mutation, domains and regions involved in chaperone ac- oxidative state, or other perturbations. Small tivity. Both the NTR and the ACD have been changes in cellular environment that cause or implicated in client binding and chaperone ac- signal stress likely lead to partially unfolded, tivity for various sHSP/client pairs. Using solid- rather than fully unfolded protein states. Such

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Mechanisms of Small Heat Shock Proteins

states may display increased exposure of hydro- 2012) and likely blocks other β4–β8 interactions, phobic residues that are the likely signals for althoughthesehavebeenlesswell-studiedtodate. sHSP binding; failure to either refold such re- The groove bump mutation enhances HSPB5 gions or engage such regions by sHSPs can lead chaperone activity against Aβ1-40, which the in- to protein aggregation. Due to the instability and vestigators attributed to reduced competition heterogeneity of such states, determination of with the CTR motif (Mainz et al. 2015), although recognition motifs within binding partners is how this mutation affects client binding and the challenging. A potential recognition element is relative affinities of the client protein, the NTR, the I/V-X-I/V motif—that is, the same motif and the CTR for the β 4–β8 groove remain to be present in sHSP NTRs and/or CTRs that binds determined. The position analogous to S135 in the β4–β8 groove of the ACD. The presence of in HSPB5 is conserved as a small sidechain in I/V-X-I/V-like motifs in putative clients would all human sHSPs, so analogous bump muta- suggest an interesting possibility of a competi- tions can easily be designed for other systems tion between sHSP subunits and clients for (Fig. 1). “GXG” mutations in the CTR of HSPB5 binding to the grooves. As mentioned earlier, and HSPB1 have been used to eliminate the the CTR I/V-X-I/V motif of HSPB5 exists in a I/V-X-I/V motif from these regions by mutating bound/unbound equilibrium and the popula- the isoleucine and valine residues to glycine, tions of the two states are dependent on con- such that the motif no longer binds the groove ditions such as temperature and pH. Therefore, (Delbecq et al. 2012). These and analogous mu- availability of the β4–β8 groove for client bind- tationsinothersHSPswillenabletheimportance ing may be modulated by environmental condi- of CTR/client competition to be assessed. tions. The client protein tau contains I/V-X-I/V Despite its clear implication in client bind- sequences within two aggregation-prone motifs ing, details regarding potential recognition ele- that are recognized by HSPB1 (Baughman et al. ments within the NTR are almost completely 2018). Although the tau-binding site on HSPB1 lacking. Whether sHSPs recognize sequence fea- has not been explicitly determined, it is predict- tures beyond exposed hydrophobic residues, ed to be the β4–β8 groove. The peptide Aβ1-40 whether there are differences between sequences and the protein α-synuclein bind in the β4–β8 bound by the ACD and by the NTR, and how the groove of HSPB5 via the sequences LVFFA and interactions function to delay protein aggrega- DVFMK, respectively (Mainz et al. 2015; Liu et al. tion are questions for the future. 2018). These sequences do not strictly follow the I/V-X-I/V motif, but are enriched in other hydro- sHSP FUNCTION phobic residues, lending further support for the notion that the definition of β4–β8groove- sHSPs are best known as early responders to binding sequences should be expanded to include cellular stress that delay the onset of irreversible other hydrophobic residues. Nonclient sHSP- protein aggregation. They are also implicated in binding partner and HSP70 cochaperone BAG3 a growing number of other processes including also binds in the groove (Rauch et al. 2017), pro- cellular signaling and regulation of apoptosis. viding an additional level of potential competitive How and whether the chaperone and nonchap- binding among cellular proteins. erone activities are related is an open question. The prevalence of I/V-X-I/V-like motifs and Given the diversity in human sHSP sequence, their predilection to bind to β4–β8 grooves of oligomerization, and tissue distribution, it is sHSP ACDs raises the important question of likely that different sHSPs have evolved to re- whether an observed interaction is functionally spond to specific forms of stress experienced relevant or serendipitous. Structure-based muta- in different tissue types (Table 1). For example, tions can be used to parse out this key question. cardiac muscle is subject to chronic stress from The “groove bump” mutation, S135Q in HSPB5, contraction and may also be subjected to acute blocks binding of the CTR I/V-X-I/V motif of stress conditions in pathological or disease con- HSPB5 to the β4–β8 groove (Delbecq et al. ditions (Lomiwes et al. 2014). Whether the same

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sHSPs are responsible for both chronic and and HSPB5 affect binding to the cytoskeleton, acute stress responses is as yet unknown. implying the importance of these interactions (Andley et al. 2014). Lens sHSPs also protect against UV-A radiation and regulate apoptosis Constitutive Roles of sHSPs through signaling. On UV-A stress, HSPB4 Constitutive roles for sHSPs include regulation activates the Akt antiapoptotic pathway and of cytoskeletal elements, cell signaling, and main- HSPB5 prevents activation of the RAF/MEK/ tenance of eye lens transparency and refractive ERK pathway (Liu et al. 2004). Thus, even in a properties (Dubińska-Magiera et al. 2014; Bak- tissue as seemingly simple as the eye lens, sHSPs thisaran et al. 2015; Carra et al. 2017). To illus- fulfill both chaperoning and nonchaperoning trate the diverse roles of sHSPs, we discuss sHSP functions. function in the eye lens and muscle tissue. sHSPs also play constitutive and stress-in- The eye lens is a specialized structure that duced roles in cardiac and skeletal muscles. transmits and focuses light on the retina. Trans- Functional myofibrils require systematic and parency and absence of light diffraction is controlled movements of cytoskeletal elements achieved by the removal of all subcellular struc- for muscle contraction. Muscles are subject to tures, including the nucleus from lens fiber cells, high oxidative stress conditions in their normal creating cells with minimal protein turnover working modes so it is perhaps not surprising ability. This situation imposes a strong require- that many sHSPs are expressed at high level in ment for protective mechanisms in these cells as different types of muscle tissue: HSPB1, HSPB2, the ability to dispose of misfolded and damaged HSPB3, HSPB5, HSPB6, HSPB7, and HSPB8 proteins is reduced and the ability to synthesize (Beall et al. 1997; Sugiyama et al. 2000; Kappé new ones does not exist (Pereira et al. 2003; et al. 2001). Specific muscle sHSPs may partly or Lynnerup et al. 2008). Lens proteins are suscep- fully diverge in function. For example, HSPB7 tible to damage caused by ultraviolet (UV) light has a role in maintaining myofiber structure and and oxidative stress, both of which lead to chem- intercalated disc integrity but shows no protec- ical modifications, yet, lens proteins must be tion against protein aggregation in cell lysate ex- maintained in soluble forms throughout the periments (Wales et al. 2016; Liao et al. 2017; life of an individual to avoid formation of in- Mymrikov et al. 2017). An emerging area of insoluble protein aggregates (i.e., lens cataract) vestigation is a role for sHSPs in the mainte- (Michael and Bron 2011; Yanshole et al. 2013; nance of cytoskeletal integrity through interac- Srivastava et al. 2017). Two members of the tions with muscle proteins, including titin, sHSP family, HSPB4 and HSPB5 (also known desmin, actin, and 14-3-3 (Mounier and Arrigo as α-crystallins), are responsible for this critical 2002; Houck et al. 2011; Diokmetzidou et al. function. HSPB4 and HSPB5 account for 40% of 2016; Sluchanko et al. 2017; Unger et al. 2017). the total protein content in the lens, where they sHSPs also act as antiapoptosis regulators and exist as soluble oligomers at concentrations protect mitochondria against stress in muscle above 150 mg/mL (Bloemendal 1977). Weak cells (Garrido et al. 1999; Kamradt et al. 2002; interactions with other crystallin proteins facili- Morrison et al. 2003; Fan 2005; Maloyan 2005; tate uniform protein distribution in the lens, Havasi et al. 2008). The growing list of processes yielding a transparent, highly refractive lens nec- in which sHSPs are directly involved implies that essary for proper vision (Fu and Liang 2002; they are responsible for a broad range of protec- Takemoto and Sorensen 2008). The lens sHSPs tive mechanisms that are central to proteostasis. serve additional functions through interactions with cytoskeletal proteins such as actin and in- Stress-Related Role of sHSP: Mechanisms termediate filaments such as vimentin, filensin, of Activation and phakinin (Nicholl and Quinlan 1994; Mu- chowski et al. 1999; Andley et al. 2014; Cheng To fulfill their protective function of delaying et al. 2017). Disease-related mutations in HSPB4 the onset of irreversible protein aggregation,

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Mechanisms of Small Heat Shock Proteins

sHSPs must be highly sensitive to small environ- associated with some disease states. In tumor mental changes and must respond quickly. development, intracellular pH is increased and Human body temperature is strongly regulated extracellular pH is decreased (Shirmanova et al. within a few degrees Celsius at most, and as little 2015; Huber et al. 2017). Acidification of brain as a 0.1 pH unit change in pH is considered tissue as a function of aging and in Alzheimer’s acidosis or alkalosis (Juel 2008). The most ex- and Parkinson’s disease patients has been re- treme acidotic pH change in exercising muscle is ported (Forester et al. 2009; Henderson et al. less than 1 pH unit, from pH 7.4 to 6.5 (on 2014; Hu et al. 2015; Majdi et al. 2016). A de- average, pH 6.9 constitutes acute muscle acido- crease in pH is also found in ischemic tissues sis) (Carter et al. 1967; Wray 1988; Street et al. with restricted blood supply (e.g., brain, heart, 2001). Here, we will discuss activation mecha- and kidneys) (Marzouk et al. 2002; McVicar et nisms of sHSPs in response to cellular condi- al. 2014; Longo et al. 2017). sHSPs are activated tions. by ischemic conditions (Martin et al. 1997; Stet- ler et al. 2012; Sharp et al. 2013). Lens cells that Thermal Activation depend on the activity of their high constitutive levels of HSPB4 and HSPB5 have a normal pH of As their name suggests, sHSPs are activated by ∼6.5, implying that these sHSPs are active at the elevated temperature. However, what consti- low end of the physiological pH range (Bassnett tutes heat shock differs among organisms, and and Duncan 1985). Thus, analogous to their different sHSPs are activated in different tem- high sensitivity to small temperature changes, perature ranges depending on their environ- the sHSPs have evolved to respond to small ment or body temperature (Haslbeck and Vier- changes in pH. ling 2015). HSP16.5 from hyperthermophiles is Histidine (His) residues are the likely key to activated between 60°C and 95°C (Bova et al. pH modulation of sHSPs as the pKa of His side- 2002), although activation temperature of the chains make them well suited to respond in the lens-specific HSPB4 in fish depends on the relevant pH range of 6.4–7.5. HSPB2-7 are en- habitat water temperature (Posner et al. 2012). riched in histidines, especially within their In temperatures normal for yeast (25°C), S. cer- ACDs, containing more than twice the average evisiae HSP26 is inactive and only shows chap- across all proteomes (Fig. 1A) (Moura et al. erone function with increased temperature 2013). At pH 6.5, HSPB5 forms expanded olig- (Haslbeck et al. 1999). Although temperature- omers by a rearrangement that is dictated by a dependent sHSP activity is well documented, single histidine residue (His104) (Rajagopal the structural basis for enhanced activity remain et al. 2015b). Paradoxically, oligomeric expan- poorly understood. A small transition is detect- sion at pH 6.5 is linked to destabilization of the ed by differential scanning calorimetry at near ACD dimer interface. A mutant that mimics the physiological temperatures (37°C–45°C) for bo- protonated His104 state, H104K-HSPB5, yields vine HSPB4 and HSPB5 purified from lens cells, HSPB5 that displays low pH properties (expand- suggesting that mammalian sHSPs undergo ed oligomers and enhanced chaperone activity) some sort of structural alteration near physio- at pH 7.5. Histidine is conserved at the analo- logical temperatures (Walsh et al. 1991). Alto- gous position in seven of ten human sHSPs (Fig. gether it is clear that sHSPs have evolved to be 1A). HSPB1 shows similar pH behavior to sensitive to small changes in temperature near HSPB5 and the corresponding His124 also acts physiological temperature. as an activation switch. The effect of pH on HSPB1 is more modest than in HSPB5, high- pH Activation lighting the diversity in function and stress response of sHSPs (Clouser and Klevit 2017). pH In mammals, even small variations in cellular activation has also been shown for an sHSP from pH can have grave consequences (Krieg et al. C. elegans, Sip1 (Fleckenstein et al. 2015). In- 2014). Furthermore, changes in cellular pH are triguingly, some Sip1 histidines, including the

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M.K. Janowska et al.

position corresponding to His104 in human many diseases (Jomova and Valko 2011; Jai- HSPB5, align with those in human sHSPs, but shankar et al. 2014). Changes at the level of pH has the opposite effect on oligomeric ensem- intracellular metal localization, rather than total ble size, with Sip1 oligomeric size decreasing metal concentration may also be important. with decreasing pH (Fig. 1A). These observa- Dysregulation of metal homeostasis is linked to tions suggest that the pH activation mechanism oxidative stress, as accumulation of metals facil- of sHSPs may use similar residues (primary/ itates formation of free radicals (Jaishankar et al. secondary structure), but with structurally di- 2014). Comprehensive information about the vergent ramifications at the level of higher order response of sHSPs to metals is not yet available organization. A more complete understanding but it is known that certain metals can induce of sHSP pH activation will require additional expression of some sHSPs. For example, Cu2+ studies on other members of the family. and Cd2+ increase expression of sHSPs in human epithelial cells transformed into lens fibers 2+ Oxidative Stress and Metals (Ganadu et al. 2004). Exposure to Cd affects the expression level of sHSPs in aquatic midges Oxidative stress occurs when reactive oxygen (Martín-Folgar and Martínez-Guitarte 2017). In species (ROS) formed in cells fail to be properly response to As3+ exposure, HSPB1 expression neutralized. This situation can occur from nor- was decreased via tumor suppressor p27 (Liu mal processes as well as pathological situations et al. 2010). Biochemically, HSPB1, HSPB4, such as bacterial infection or exposure to metals and HSPB5 bind Cu2+ with picomolar affinity, (Finkel 2011; Sies et al. 2017). Among the groups and a single HSPB5 oligomer can reportedly se- targeted by ROS are oxidation-sensitive amino quester up to 150 Cu2+ ions, acting as an ion acid residues in proteins whose modification can “sponge” (Prabhu et al. 2011; Mainz et al. cause changes in protein structure that can affect 2012). Cu2+ binding by the HSPB5 ACD is re- function and/or lead to aggregation (Reichmann ported to affect monomer–dimer equilibrium, et al. 2018). Surprisingly, little is known about which in turn promotes formation of larger olig- the role of sHSPs in cell protection under oxida- omers (Mainz et al. 2012). The link between tive stress. HSPB1 and HSPB5 are proposed to ACD dimer interface stability and oligomer be modulators of glutathione levels and to be size is reminiscent of that observed for the pH protective against oxidative stress in cell lines effect in HSPB5 and may point to shared or (Préville et al. 1999; Arrigo et al. 2005; Christo- overlapping effects of pH and metal ions. Zn2+ pher et al. 2014). HSPB4 and HSPB5 protect is also reported to affect sHSP ensembles (Ga- against oxidative stress in the eye lens (Wang nadu et al. 2004; Biswas and Das 2008; Karma- and Spector 1995). Structural and mechanistic kar and Das 2011). In contrast to other heavy information on sHSPs under oxidative stress is metals, zinc counteracts oxidative stress. Cur- lacking. An oxidation-sensitive cysteine located rently, there appears to be conflicting informa- at the center of the HSPB1 dimer interface may tion regarding the role of zinc in the eye lens. govern monomer–dimer exchange (Rajagopal Zinc has been shown to induce aggregation of et al. 2015a). Although a role for sHSPs in oxi- another lens protein, γD crystallin, and zinc lev- dative stress mitigation seems sensible, strong els in cataractous lenses are increased despite and compelling evidence in support of this pu- studies suggesting that zinc supplementation tative role remains missing. may protect against cataract development (Ke- Metal ions mediate many cellular processes, tola 1979; Ciaralli et al. 2001; Dawczynski et al. including oxygen utilization, immunological sys- 2002; Quintanar et al. 2016; Domínguez-Calva tem response, and enzymatic activities (Bleack- et al. 2018). Similar to the effects of Cu2+,Zn2+ ley and MacGillivray 2011). Cellular metal con- alters HSPB5 oligomer organization, stability, centrations are tightly regulated: Accumulation and chaperone function (Prabhu et al. 2011; Bis- of a given metal outside its functional concen- was et al. 2016). The proposed binding site for tration range can be toxic and is implicated in Zn2+ is located within the ACD (Mainz et al.

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Mechanisms of Small Heat Shock Proteins

2012). The pH and metal ion effects described phosphomimics show decreased chaperone to date suggest that the ACD is responsible not activity toward ccβ-Trp, luciferase, and LDH, only for forming the dimeric building block of and phospho-HSPB1 has decreased activity oligomers, but also in controlling oligomeric toward citrate synthase (Rogalla et al. 1999; Ito size and dynamics in ways yet to be fully defined. et al. 2001; Ecroyd et al. 2007). Moreover, it appears that the extent of phosphorylation is Phosphorylation important: A single phosphomimicking mutation in HSPB5 shows antiapoptotic activity (in- In addition to the direct structural and function- hibition of caspase-3), although the triple phos- al responses of sHSPs to cellular conditions, phomimic does not (Morrison et al. 2003). their activity is regulated by phosphorylation. In summary, sHSPs are highly sensitive to Phosphorylation of sHSPs has been implicated small changes in cellular environment and can in the regulation of multiple cellular functions respond rapidly to change. Changes in condi- including apoptosis, cytoskeletal modulation, tionssuchaspH,temperature,andmetalbinding cell-cycle regulation, ligand binding, and chap- bring about changes in sHSP structure and, erone activity and is implicated in disease con- therefore, activity. Their adaptability is mediated ditions. Hyperphosphorylated HSPB1 is found through hierarchical structural organization and, with tau in neurofibrillary tangles and hyper- possibly, changes in their dynamics that involve phosphorylated HSPB5 mutant, R120G, accu- both ordered and disordered regions of sHSPs. mulates in insoluble fractions (Nemes et al. However, although a boon to their cellular func- 2004; Shimura et al. 2004; Bakthisaran et al. tions, the intrinsic plasticity of sHSPs render 2016). The kinases involved in sHSP regulation their rigorous study challenging, as small alter- are known in some instances, but detailed infor- ations in experimental conditions can have pro- mation about all possible players in the regula- found effects on their properties and function. tion of sHSPs is yet to be determined. Most phosphorylation sites identified in sHSPs are MECHANISMS OF CLIENT INTERACTIONS localized in the disordered NTR where the modification likely exposes binding sites that provide A central question in the sHSP field is: How are enhanced chaperone activity and additional ho- clients recognized and engaged? Historically, meostatic functions. There are three sites in the studies have tended to use model client proteins NTRs of HSPB1 and HSPB5 (15, 78, and 82 and such as α-lactalbumin and κ-casein, as they pro- 19, 45, and 59, respectively). Phosphorylation is vide experimentally tractable systems (Ecroyd often mimicked experimentally by substitution et al. 2007; Kulig and Ecroyd 2012). More re- of the serine residues that are phosphorylated cently, bona fide clients known to aggregate with negatively charged aspartate or glutamate. within the cell, such as the amyloid-forming Studies on phosphorylated or phosphomimics proteins tau, Aβ, and α-synuclein, and metabol- of HSPB1 and HSPB5 reveal a substantial de- ic enzymes that form amorphous aggregates un- crease in the average oligomer size relative to der destabilizing conditions have been investi- unphosphorylated protein, with fully phosphor- gated (Mainz et al. 2015; Cox et al. 2016; ylated species existing predominantly as dimers Mymrikov et al. 2017; Baughman et al. 2018; or tetramers of HSPB1 or 6-mers and 12-mers of Liu et al. 2018). Important open questions in- HSPB5 (Rogalla et al. 1999; Peschek et al. 2013; clude what regions of sHSPs are involved in cli- Jovcevski et al. 2015). In most but not all in- ent binding and chaperone activity, are there stances, phosphorylation (or its mimicry) is as- common features in clients that enable sHSP sociated with an increase in chaperone activity recognition, what types of client/sHSP complex- toward clients (Koteiche and McHaourab 2003; es are formed and what are the species of aggre- Shashidharamurthy et al. 2005; Meehan et al. gation-prone clients that are effectively engaged 2007; Ahmed et al. 2009; Hayes et al. 2009; Pe- by the sHSP, and how do sHSP oligomerization schek et al. 2013; Jovcevski et al. 2015). HSPB5 and subunit exchange dynamics relate to chap-

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M.K. Janowska et al.

erone activity. The current state of knowledge proteins, emphasizing the sensitivity of results regarding the nature and identity of sHSP/client to the specific sHSP/client pair studied and the interaction sites was presented in the section on perils of attempting to generalize results. As al- Structural Features of sHSP/Client Interactions. ready mentioned, further complications arise Below, we review information relevant to the from the sensitivity of sHSP structure and func- other questions posed. tion to environmental conditions such as pH It is becoming clear that the types of sHSP/ and temperature. Two additional confounding client complexes formed vary considerably de- issues are raised by the fact that sHSPs can as- pending on the sHSP, the client, the conditions, sociate with each other and exist as hetero-olig- and the client aggregation pathway and type of omeric species in cellular contexts and that aggregate formed. HSPB5 and HSPB1 interact sHSPs are not presented with one pure client weakly and transiently with the clients Aβ, tau, under cellular stress situations, but rather must and α-synuclein, as evidenced by the inability engage and delay aggregation for many cellular to detect complexes by NMR or size-exclusion proteins simultaneously. These important issues chromatography (Mainz et al. 2015; Cox et al. remain to be addressed in the future. 2016; Baughman et al. 2018). Notably, these The oligomeric state of sHSP/client com- clients are intrinsically disordered in solution plexes and the rate at which subunits exchange and form amyloid fibrillar aggregates. In con- in and out of oligomers also influence chaperone trast, when presented with α-synuclein fibrils, activity, but there is no consensus on the role of HSPB1 was shown to form a tight complex with these factors. Some have argued that the chap- mature fibrils but not with prefibrillar species erone-active sHSP species are small oligomers or (Cox et al. 2018). Similarly, HSPB5 coprecipi- dimers, and that reduced oligomeric size and tates with aggregated lysozyme, which forms rapid subunit exchange of these species enables amorphous aggregates (Mainz et al. 2015). To enhanced chaperone activity (Peschek et al. test whether HSPB5 interacts differently with 2013; Jovcevski et al. 2015). However, others an amyloid-forming versus amorphously aggre- have documented diminished chaperone activi- gating client, Kulig and Ecroyd (2012) investi- ty from phosphorylated sHSPs that form small gated the mechanisms by which HSPB5 inhibits oligomers and have argued that larger oligomers amorphous aggregation of reduced α-lactal- are the chaperone-active species (Rogalla et al. bumin and amyloid formation of reduced and 1999). Again, the specific effect is likely depen- carboxymethylated α-lactalbumin under other- dent on the sHSP/client pair studied and the wise-similar experimental conditions. They conditions under which chaperone activity is found that HSPB5 forms a stable complex with assessed. For example, under acidosis condi- α-lactalbumin under conditionsthat promote its tions, HSPB5 forms enlarged oligomers. A low amorphous aggregation but interacts only tran- pH-mimicking mutation H104K-HSPB5, forms siently with α-lactalbumin to delay the onset of similarly enlarged oligomers under “normal” amyloid formation. Although it is tempting to pH conditions and has enhanced chaperone generalize these observations to suggest that activity toward destabilized α-lactalbumin (Ra- chaperone activity against amyloid-forming cli- jagopal et al. 2015b). Although the wild-type ents occur via transient interactions, whereas (unactivated) HSPB5 interacts with the clients sHSPs delay formation of amorphous aggregates via only weak, transient interactions, the acti- by forming more stable complexes, it is unlikely vated form disassembles into smaller species that this simple relationship will hold once more that coelute with client on size exclusion chro- data are in hand. matography. Thus, the precise details of how a Another emerging theme is that different given client’s aggregation is delayed by an sHSP sHSPs may interact with the same client protein is a complicated and interconnected process in through distinct mechanisms. Mymrikov et al. which factors such as oligomerization and sub- (2017) showed that different human sHSPs dis- unit exchange dynamics, binding site accessibil- play differential activities against various client ity, affinity for client protein, mechanism of

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Mechanisms of Small Heat Shock Proteins

aggregation and the nature of client species these standard aggregation assays provide a sim- formed along the pathway all contribute (de- ple way to assess the ability of an sHSP to delay picted in Fig. 3). This likely gives rise to the the onset of and/or inhibit aggregation, but there enormous diversity and adaptability of sHSP are important limitations. In particular, the function, but also makes it nearly impossible strength of the light-scattering signal is a func- to determine the influence each has on chaper- tion of both the size and amount of aggregates one activity independently of other factors. being formed (Den Engelsman et al. 2011), al- In summary, recent studies have revealed though thioflavin T fluorescence is sensitive to ways in which sHSPs accomplish chaperone ac- both fibril mass and morphology (Lindberg et al. tivity. The mechanisms of action documented 2015)—a complication further confounded by thus far depend on the specific sHSP/client the fact that these properties will be changing pair under consideration and on the client’s with time and may be dependent on differences mechanism of aggregation, suggesting that in salt, pH, temperature, or other conditions. sHSP chaperone activity may not be fully de- Therefore, while standard aggregation assays scribed by a single unified model. Rather, it high- are both widely performed and extremely infor- lights the breadth of function present within this mative, they are best used as qualitative and class of chaperones as they target diverse cellular comparative assessments of chaperone activity. clients. That said, they are the most robust and most experimentally accessible approach for assessing sHSP chaperone activity in vitro. Studies aimed EXPERIMENTAL CHALLENGES AND THE at characterizing effects of a perturbation (mu- FUTURE tation, change in environmental condition, etc.) Much of our current understanding of the on sHSP function will benefit from the use of mechanisms of sHSP chaperone function is multiple client proteins, and use of uniform ex- based on in vitro assessment of function using perimental conditions. client proteins that can be selectively destabi- A study in which chaperone activity of all ten lized to produce aggregates (Fig. 3). Two over- human sHSPs was assessed with several differ- arching goals of in vitro assessment of sHSPs are ent model clients under different destabilization to (1) determine “all” underlying mechanisms conditions provides both valuable information by which sHSPs function, and (2) define “spe- and a cautionary tale regarding the level of con- cific” mechanisms by which a given sHSP works trol required to carry out experiments that can with a specific client. Both are important and be compared across systems (Mymrikov et al. these two goals require different approaches. 2017). Not only were differences in activity to- The workhorse experiment of in vitro chaperone ward a given client observed among sHSPs, but studies is the aggregation assay in which the ef- differences in client aggregation that depend on fect of presence or absence of an sHSP on a client solution conditions were also observed. This im- aggregation time course is assessed. Clients dif- portant study emphasizes the hazards of draw- fer in their requirements for destabilization and ing general conclusions on sHSP function based aggregate morphology. Aggregation of com- on the use of a single client or experimental con- monly used model clients such as α-lactalbu- dition and adds an additional level of complexity min, lysozyme, and β-crystallin is initiated by to efforts to translate in vitro findings on sHSP reduction and/or increased temperature. The function to a cellular setting. Thus, while model appearance of aggregates is most often moni- clients such as α-lactalbumin have and will con- tored by measuring light scattering, usually de- tinue to provide important insights into funda- tected as increased absorbance of light at 360 nm mental aspects of sHSP activity, identification fi (A360nm), as a function of time. Aggregation of and characterization of bona de cellular clients clients that form amyloid-type fibrils such as tau are important directions for the future. In addi- is typically monitored by thioflavin T fluores- tion, assessment of sHSP properties such as olig- cence (Biancalana and Koide 2010). Overall, omer size and distribution, subunit exchange

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Heat shock, pH acidification, metal binding

Phosphorylation

sHSP ensemble

Client

Aggregation Native prone Intermediate Aggregate

Figure 3. Diversity of small heat shock protein (sHSP) chaperone mechanisms. Many human sHSPs form poly- disperse oligomers whose average number of subunits is sensitive to solution conditions or posttranslational modiﬁcations. Client aggregation is also sensitive to solution conditions and can potentially affect the client states recognized by an sHSP. It is unknown whether all subunits within a given sHSP ensemble are chaperone active. Many open questions remain, including: (1) Which client states and features of clients do sHSPs recognize? (2) Do different sHSP ensemble sizes recognize different client states and therefore have different chaperone activity? The answers to these and other questions likely depend on sHSPs and client identities and the solution conditions used to assess chaperone function.

rate, time required for an sHSP to equilibrate to a itsinfancy.Thesefascinatingproteinshavemost- given condition, availability of client binding ly defied biochemical and structural analysis for surfaces, and dynamics under the chosen exper- reasons discussed above and this has, in turn, imental conditions will greatly improve our abil- slowed progress in understanding the molecular ity to interpret the results. As the biochemical and cellular biologyofsHSPs. Asstructure-based understanding of sHSP function progresses, ex- and mechanism-based sHSP mutants are devel- perimental designs that aim for more “cell-like” oped (e.g., the groove-bump mutant discussed conditions are needed. These could include mix- earlier), these will provide powerful tools with tures of sHSPs in ratios that reflect those in a which to investigate the mechanisms of action given cell. Of course, even this “simple” param- of sHSPs in cells. Furthermore, emerging tech- eter is likely to depend on both cell type and cell nological advances and approaches more suited conditions. Similarly, increasing the complexity to dynamic heterogeneous systems promise to be of the “client” from a single, purified (model) transformational to the field. Application of hy- client protein to mixtures or to cell lysates could drogen–deuterium exchange, single-molecule provide additional layers of insight. approaches, native mass spectrometry, cryo- Despite progress made over the past several EM, and solid- and solution-state NMR afford decades, we believe that sHSP research is still in exciting possibilities. Coupled with cellular ap-

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Mechanisms of Small Heat Shock Proteins

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Mechanisms of Small Heat Shock Proteins

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Mechanisms of Small Heat Shock Proteins

Maria K. Janowska, Hannah E.R. Baughman, Christopher N. Woods and Rachel E. Klevit

Cold Spring Harb Perspect Biol published online March 4, 2019

Subject Collection Protein Homeostasis

Proteome-Scale Mapping of Perturbed The Amyloid Phenomenon and Its Significance in Proteostasis in Living Cells Biology and Medicine Isabel Lam, Erinc Hallacli and Vikram Khurana Christopher M. Dobson, Tuomas P.J. Knowles and Michele Vendruscolo Pharmacologic Approaches for Adapting A Chemical Biology Approach to the Chaperome Proteostasis in the Secretory Pathway to in Cancer−−HSP90 and Beyond Ameliorate Protein Conformational Diseases Tony Taldone, Tai Wang, Anna Rodina, et al. Jeffery W. Kelly Cell-Nonautonomous Regulation of Proteostasis Proteostasis in Viral Infection: Unfolding the in Aging and Disease Complex Virus−Chaperone Interplay Richard I. Morimoto Ranen Aviner and Judith Frydman The Autophagy Lysosomal Pathway and The Proteasome and Its Network: Engineering for Neurodegeneration Adaptability Steven Finkbeiner Daniel Finley and Miguel A. Prado Functional Modules of the Proteostasis Network Functional Amyloids Gopal G. Jayaraj, Mark S. Hipp and F. Ulrich Hartl Daniel Otzen and Roland Riek Protein Solubility Predictions Using the CamSol Chaperone Interactions at the Ribosome Method in the Study of Protein Homeostasis Elke Deuerling, Martin Gamerdinger and Stefan G. Pietro Sormanni and Michele Vendruscolo Kreft Recognition and Degradation of Mislocalized Mechanisms of Small Heat Shock Proteins Proteins in Health and Disease Maria K. Janowska, Hannah E.R. Baughman, Ramanujan S. Hegde and Eszter Zavodszky Christopher N. Woods, et al. The Nuclear and DNA-Associated Molecular Structure, Function, and Regulation of the Hsp90 Chaperone Network Machinery Zlata Gvozdenov, Janhavi Kolhe and Brian C. Maximilian M. Biebl and Johannes Buchner Freeman

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